As evidenced by proteomics analyses, human platelets contain a myriad of molecules exhibiting important physiological functions, among which growth factors (GF) are of particular interest. These growth factors, which accumulate within the platelet α-granules, mainly include three Platelet-Derived Growth Factors isoforms (PDGF-AA, -AB, and -BB), Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-β (TGF-β1 and TGF-β2), Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and some Insulin-like Growth Factor (IGF). According to the therapeutic interest of such growth factors, complex human platelet growth factors mixtures are already being used for therapeutic applications in the form of single-donor platelet gel biomaterials resulting from the activation of platelet-containing plasma by thrombin.
Platelet activation generally leads to the release of the growth factors contained therein and their entrapment into a gel matrix obtained by simultaneous thrombin-induced conversion of fibrinogen into fibrin. This biomaterial is generally applied on tissues, alone or in combination with graft materials. Growth factors-containing platelet gels are thus used in regenerative medicine to promote the healing and regeneration of soft and hard tissues. The growth factors concentrates obtained from platelets are also used for supplementing the growth media for in vitro or ex vivo cell cultures. Recently, the possibility to use human growth factors-rich platelet releasates as substitute for fetal bovine serum (FBS) for ex-vivo expansion of mesenchymal or hematopoietic stem cells has been demonstrated.
The use of growth factors-rich platelets releasates is of particular interest for expanding stem cells, and more specifically Mesenchymal stem cells (MCS) (or hematopoietic stem cells) which are considered as emergent “universal” cells, as a substitute for Fetal Bovine Serum (FBS) or Fetal Calf Serum (FCS) which are considered as possible sources of prion and virus transmission, as well as a cause for immunological problems in patients due to residual bovine proteins and antigens within the Bovine or Calf serum.
New approaches were recently developed, consisting in the production of recombinant platelet growth factors in conventional expression systems. For instance, some recombinant individual human (Rh) growth factors are used for therapeutic applications. Rh-PDGF-BB expressed by genetically-engineered yeast is in particular licensed in some countries for the treatment of chronic neuropathic lower-extremity diabetic ulcers although large doses and several weeks application of this single growth factor appear needed to achieve clinical efficacy. Similarly, Rh-bone morphogenetic proteins (BMP)-2 and -7, which belong to the TGF-β superfamily, can be produced from Chinese Hamster Ovary cells, and are licensed to heal some fracture surgery. However, the number of available recombinant growth factors remain extremely limited, which is in part due to the difficulty to isolate, identify, clone and express these growth factors. Furthermore, platelet-derived preparations still provide a supplementary benefit over the use of single recombinant factors, since a beneficial synergistic effect of growth factors combination can be achieved in some applications.
It nevertheless appears that the growth factors PDGF and VEGF were shown to be directly linked to the abnormal propagation and/or differentiation of cells (see for instance Ferrara et al., Nature Medicine, vol. 9, no. 6, 2003, pp. 669-676; as well as in the publication EMA/92326/2010 of the European Medical Agency dated Feb. 18, 2010, and in the communication of the FDA dated Mar. 27, 2008 about “an ongoing safety review of regranex (becaplermin). In view of the increased susceptibility of developing cancers upon treatment with each of these two growth factors, PDGF and VEGF are thus to be avoided for culturing stem cells or for healing patients, in particular when said patients are known to be at risk of cancer.
There is therefore a need for developing processing methods of human platelet materials allowing the preparation of well-characterized virally-safe industrial growth factors preparations, and more particularly of preparations depleted in PDGF and VEGF.
Patent Application WO 2009/087560 discloses that human platelet concentrates can be subjected to a treatment by a combination of solvent and detergent (S/D) that achieves dual purposes: (a) the inactivation of lipid-enveloped viruses and (b) the massive release of the growth factors from the platelet α-granules. A standardized complex platelet growth factors mixture comprising PDGF-AB, -BB, and -AA, TGF-β, EGF, VEGF, and insulin-like growth factor-β1 (IGF-β1), could then be obtained by combining S/D treatment, oil extraction and hydrophobic interaction chromatography (HIC), wherein oil extraction and HIC were used to remove the S/D agents without affecting significantly the PGF and protein content. The resulting platelet growth factors preparation was shown to stimulate various cell lines in vitro, and enhanced the expansion of stem cells from lipo-aspirated fat tissue. In addition, WO 2009/087560 discloses that replacing the HIC by a SP-sepharose cation-exchange column allows the preparation of a purified PDGF-VEGF fraction. In this approach, PDGF and VEGF are indeed adsorbed on the column but TGF-β, EGF, IGF are found in the breakthrough, together with the S/D agents used for the virus inactivation step. Conversely, WO 2009/087560 discloses that TGF-β and EGF may be purified to a certain extent by replacing the HIC by a DEAE-sepharose-anion-exchanger.
In the publication El-Ekiaby et al. (El-Ekiaby et al; “Solvent-detergent filtered (S/D-F) fresh frozen plasma and cryoprecipitate minipools prepared in a newly designed integral disposable processing bag system”, Transfusion medicine, 2010, 20, 48-61), the authors disclose a process applied for the viral inactivation treatment of singulet or mini-pools of plasma and cryoprecipitate for transfusion and comprising, as major steps, the S/D treatment of Fresh Frozen Plasma (FFP) or of Cryo-Poor Plasma (CPP), or of cryoprecipitate with a mixture of TnBP and TRITON X-45®, the oil extraction of the S/D treated plasma material, and the filtering of the oil-extracted plasma material through an S/D adsorption filter containing activated charcoal. As discussed in this publication, the disclosed method in particular preserves the functional activity of plasma proteins (such as clotting factors), does not alter the VWF multimer composition, reduces TnBP and TRITON X-45® below acceptable levels, and allows the sterile filtration of the resulting purified plasma by gravity. This publication is nevertheless exclusively directed to plasma material purification and thus fails to disclose or even suggest a method which could be implemented for purifying growth factors from platelet concentrates.
The present inventors have now found surprisingly and after intensive research, that a process comprising a S/D treatment of a platelet concentrate, an oil extraction and a charcoal extraction, or a charcoal extraction only, permits the easy, rapid and efficient preparation of a virally inactivated growth factors containing platelet lysate which is depleted in both PDGF and VEGF, and wherein solvent and detergent concentrations are meeting the limits approved by regulatory authorities for S/D-treated parenteral blood-derived therapeutic products.
In particular, the method of the invention does not modify the content in the major proteins, such as albumin and immunoglobulins, and has essentially no impact on the concentration of growth factors others than PDGF and VEGF, such as TGF-β1, EGF, and IGF.
Further, the method of the invention allows the dissolution of lipid membranes, therefore inactivating lipid-enveloped viruses, and other pathogens like, for instance, bacteria and parasites such as protozoae, as well as the removal of plasma and platelet lipids which are present in the starting platelet concentrate. The method of the present invention thus renders it possible to provide virally-inactivated platelet-derived growth factors mixtures, which could be efficiently standardized for use in therapeutic treatments, cell therapy or cell culture.
Finally, considering the fact that, in part due to the risk of bacterial contamination and loss of functional activity for hemostasis, platelets usually have a limited shelf life of 5 or 7 days, when clinically used intravenously for the correction of quantitative or functional thrombocytopenia, a high number of platelet units older than 5 or 7 days are usually discarded each year. In allowing expired platelets stocks to be used for the preparation of platelet-derived concentrates, the method of the invention finally reveals an extremely promising economical interest.